When glucose (sugar) is present in high levels in cells, it is re-directed into a pathway that leads to the stimulation of an enzyme which is responsible for the addition of 2-N-acetylglucosamine (O-GlcNAc) to over 1,000 proteins in the cell. The correct activity of this enzyme (O-GlcNAc transferase or OGT) is critical for controlling the ability of a cell to respond to its environment and prevent aberrant growth and proliferation. The function of this enzyme has been linked to proper insulin signaling and diabetes, cancer and Alzheimer's disease. Recently it has been shown that the activity of OGT is increased in breast cancer cells. Reduction of enzyme levels and its activity by either biochemical methods or an OGT-specific inhibitor lead to an inhibition of tumor growth and decreased cancer cell invasion. The long term goal of the proposed is to characterize the mechanism of OGT function; details of which are critical for the treatment of the cellular processes and diseases it has been linked to. The objectives of this proposal are to 1) develop a biochemical model of OGT function 2) search for OGT substrates using protein microarrays and 3) examine the role of OGT as a peripheral membrane binding protein. In order to develop a biochemical model of OGT function, the recently solved crystal structure of the enzyme will be utilized to mutate regions of the protein that affect its ability to properly recognize its cellular targets. With this information, much can be learned about the mechanism that OGT utilizes to differentially glycosylate proteins in the cell. To expand the knowledge of the cellular targets (and the processes those targets are responsible for) of OGT, a microscopic array that contains over 9000 human proteins will be incubated with OGT and its mutants. After detection of the proteins that have been glycosylated, a comprehensive map of OGT targets can be built. This map can be used, in the future, to selectively inhibit glycosylation of a subset of OGT targets. During insulin signaling, OGT is effectively recruited to the inner membrane of cells where it acts on members of the insulin signaling pathway. The mechanism of OGT's recruitment / binding to the cell membrane remains poorly understood. A series of biochemical and cell-based methods will be employed to determine the particular membrane components that OGT binds to and which region of the enzyme is responsible for this action. Taken together, the objectives in this proposal and the methods used to achieve them will greatly enhance our knowledge of OGT's glycosylation mechanism.